title: Crystal Structure of Crataeva tapia Bark Protein (CrataBL) and Its Effect in Human Prostate Cancer Cell Lines.
abstract: A protein isolated from the bark of Crataeva tapia (CrataBL) is both a Kunitz-type plant protease inhibitor and a lectin. We have determined the amino acid sequence and three-dimensional structure of CrataBL, as well as characterized its selected biochemical and biological properties. We found two different isoforms of CrataBL isolated from the original source, differing in positions 31 (Pro/Leu); 92 (Ser/Leu); 93 (Ile/Thr); 95 (Arg/Gly) and 97 (Leu/Ser). CrataBL showed relatively weak inhibitory activity against trypsin (Kiapp = 43 µM) and was more potent against Factor Xa (Kiapp = 8.6 µM), but was not active against a number of other proteases. We have confirmed that CrataBL contains two glycosylation sites and forms a dimer at high concentration. The high-resolution crystal structures of two different crystal forms of isoform II verified the β-trefoil fold of CrataBL and have shown the presence of dimers consisting of two almost identical molecules making extensive contacts (∼645 Å(2)). The structure differs from those of the most closely related proteins by the lack of the N-terminal β-hairpin. In experiments aimed at investigating the biological properties of CrataBL, we have shown that addition of 40 µM of the protein for 48 h caused maximum growth inhibition in MTT assay (47% of DU145 cells and 43% of PC3 cells). The apoptosis of DU145 and PC3 cell lines was confirmed by flow cytometry using Annexin V/FITC and propidium iodide staining. Treatment with CrataBL resulted in the release of mitochondrial cytochrome c and in the activation of caspase-3 in DU145 and PC3 cells.
authors.affiliation: Departamento de Bioquímica, Universidade Federal de São Paulo, São Paulo, São Paulo, Brazil.
authors.forename: Rodrigo da Silva
authors.initials: Rda S
authors.lastname: Ferreira
authors.forename: Dongwen
authors.initials: D
authors.lastname: Zhou
authors.forename: Joana Gasperazzo
authors.initials: JG
authors.lastname: Ferreira
authors.forename: Mariana Cristina Cabral
authors.initials: MC
authors.lastname: Silva
authors.forename: Rosemeire Aparecida
authors.initials: RA
authors.lastname: Silva-Lucca
authors.forename: Reinhard
authors.initials: R
authors.lastname: Mentele
authors.forename: Edgar Julian
authors.initials: EJ
authors.lastname: Paredes-Gamero
authors.forename: Thiago Carlos
authors.initials: TC
authors.lastname: Bertolin
authors.forename: Maria Tereza
authors.initials: MT
authors.lastname: Dos Santos Correia
authors.forename: Patrícia Maria Guedes
authors.initials: PM
authors.lastname: Paiva
authors.forename: Alla
authors.initials: A
authors.lastname: Gustchina
authors.forename: Alexander
authors.initials: A
authors.lastname: Wlodawer
authors.forename: Maria Luiza Vilela
authors.initials: ML
authors.lastname: Oliva
mesh_terms: D000595:Amino Acid Sequence; D031483:Capparaceae / Q000737:chemistry*; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D018360:Crystallography, X-Ray; D019281:Dimerization; D004354:Drug Screening Assays, Antitumor; D006031:Glycosylation; D006801:Humans; D008297:Male; D015394:Molecular Structure; D037121:Plant Lectins / Q000737:chemistry / Q000494:pharmacology*; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: C000598243:CrataBL; D037121:Plant Lectins